Sigma receptors are saturable, high affinity binding sites for several important classes of psychotropic drugs, including typical antipsychotic, antidepressant, anticonvulsant, and psychotomimetic compounds. They are likely to contribute to the beneficial and/or side-effect profile of these compounds. Two pharmacologically defined subtypes are known to exist, termed sigma-1 and sigma-2. Sigma sites are present not only in the central nervous system but also occur in several peripheral tissues, and are expressed in high density in a number of tumor cell lines. Furthermore, sigma-2 receptors are more highly expressed in rapidly proliferating cells than in quiescent cells. They may thus play an important role in cell function. Our studies have shown that agonists at sigma-2 receptors induce morphological changes, followed by apoptotic cell death. These effects were found to occur in various tumor cell lines, as well as in primary cultures of neuronal cells. Sigma-2 agonists induce both a transient and a sustained rise in intracellular calcium levels in human SK-N-SH neuroblastoma cells, which may play a role in cell proliferation or in the induction of apoptosis. The calcium is released from intracellular stores, and is apparently independent of IP3. Furthermore, sigma-2 receptor-induced apoptosis in breast tumor cell lines was found to occur by a novel pathway which was both p53- and caspase-independent. In the last reporting period, we investigated the involvement of sphingolipids in the apoptotic pathway induced by sigma-2 agonists. Ceramides and lysosphingolipids are involved in regulation of cell proliferation. Sigma-2 agonists were found to induce a rise in ceramide and sphingosylphosphorylcholine (a lysosphingolipid) with a concomitant decrease in sphingomyelin. This was not due to activation of sphingomyelinase, but appears to be due to an increase in sphingolipid-ceramide N-deacylase (SCDase)-like activity. In the current funding period, we continued to investigate the role of sphingolipids in sigma-2 receptor signaling. In collaboration with Dr. Robert Bittman (Queens College of New York) we investigated the effects of various novel ceramide analogs on the viability of tumor cells vs. normal cells and found that some analogs show cytotoxic selectivity against drug-resistant breast tumor cells compared to normal breast epithelial cells. This could have implications for selective induction of apoptosis in tumor cells in response to sigma-2 agonists. We also initiated studies involving the effect of sigma-2 agonists on HIV infection of lymphocytes. These studies were based on the observation that receptors for HIV entry are sequestered in lipid rafts, membrane microdomains that are known to sequester specific proteins. Our collaborator, Dr. James Hildreth (Johns Hopkins University), had previously shown that infection could be blocked by disruption of these membrane domains upon removal of cholesterol. Lipid rafts are composed of both cholesterol and sphingomyelin. Since sigma-2 receptor activation causes a decrease in membrane sphingomyelin, we investigated the effect of sigma-2 ligands on HIV infection of H9 lymphocytes. Treatment of H9 lymphocytes with low doses of various sigma-2 agonists caused a significant decrease in cellular sphingomyelin and a concomitant decrease in the ability of HIV to infect these cells. This suggests that sigma-2 receptor agonists may be useful agents in HIV infection. We have continued our program of sigma receptor ligand development and characterization, with particular focus this period on interaction of sigma-1 receptors with the rewarding and toxic effects of cocaine. Previous data suggests that antagonism at the sigma-1 receptor is responsible for the blockade of cocaine-induced convulsions and lethality by sigma receptor ligands. In collaboration with Dr. Andrew Coop (University of Maryland) and Dr. Rae Matsumoto (University of Oklahoma), we characterized a series of 1,4-dibenzylpiperazines as sigma receptor ligands. These compounds generally showed high affinity for sigma-1 receptors and moderate affinity for sigma-2 sites. These compounds blocked the convulsive effects of cocaine in Swiss Webster mice. However, unlike series characterized previously, the potency of these compounds tended to correlate better with sigma-2 receptor affinity than with sigma-1 affinity. This suggests the possibility that sigma-2 sites may also play a role in this effect. In collaboration with Dr. Amy Newman (NIDA) we characterized a series of novel piperazinyl alkyl-bis-(4?-fluorophenyl)amine analogs as potential cocaine-abuse therapeutics. These compounds are rimcazole analogs which exhibit affinity at both the sigma-1 receptor and the dopamine transporter. These studies are based on the observation that rimcazole, a dual sigma-1 receptor antagonist that binds to the dopamine transporter, lacks behavioral similarity to cocaine yet attenuates some of the behavioral actions of this drug. Three compounds were identified as promising for further study. In collaboration with Dr. Tangui Maurice (CNRS) and Dr. Remi Martin-Fardon (Scripps Institute) we investigated the interaction of sigma-1 receptors and neurosteroids and their effects on cocaine reward. The neuroactive steroids DHEA, pregnenolone, and progesterone are sigma-1 receptor ligands, where DHEA and pregnenolone are agonists and progesterone is an antagonist. Pretreatment of C57BL/6 mice with pregnenolone or DHEA during conditioning with cocaine, enhanced cocaine-induced conditioned place preference (CPP), while progesterone blocked it. The sigma-1 receptor antagonist, BD1047, blocked cocaine-induced CPP and its potentiation by pregnenolone and DHEA. Progesterone blocked cocaine-induced CPP and its potentiation by the sigma-1 receptor agonist, igmesine. These data suggest that neurosteroids modulate cocaine's rewarding effects via interaction with sigma-1 receptors. These results have implications for neuroendocrine control of cocaine addiction.